Many members of the genus Flavivirus are significant human pathogens. The 5'end of flavivirus plus- sense RNA genome contains a type 1 cap (m7GpppAmG). We recently found a general mechanism that the flavivirus NS5 methylates the guanine N7 on the cap and ribose 2'-OH on the first transcribed nucleotide. We also found that the N7 methyltransferase (MTase) is essential for flavivirus replication. Understanding the mechanism of cap methylation is critical for development of inhibitors of this enzyme for antiviral intervention. Our long-range goal is to study the molecular mechanisms of flaviviral replication and to develop anti-WNV therapeutics. The objective of this application is to study the molecular mechanism of flavivirus MTase. We will use West Nile Virus (WNV) as a model to accomplish three aims. Aim 1 is to define the "repositioning" mechanism of the WNV RNA cap methylation. Because of the nature that the two N7 and 2'-O methylations are sequential by one MTase, we hypothesize that substrate GpppA-RNA should be "repositioned" to accept the N7 and 2'-O methyl groups from the methyl donor, S-Adenosyl-Methionine (SAM), during the two sequential methylations. We will test the hypothesis by determining why and how the flavivirus MTases catalyze the sequential reactions. Three models including a translocation model, a dissociation-and-reassociation model, and a dimerization model will be examined. The molecular details during the switch of the two methylation events will be defined. Aims 2 and 3 are to determine crystal structures of favivirus MTase in complex with an N7 viral RNA substrate (Aim 2) and with a 2'-O viral RNA substrate (Aim 3). We recently found that flavivirus MTase methylates cap structures with distinct specificities for viral RNA sequences for the N7 and 2'-O methylations, respectively. Under Aims 2 and 3, we will crystallize and solve the structures of the WNV MTase in complex with viral RNA substrates (including both N7 and 2'-O substrates) and analog methyl donor molecule. The co-crystal structure will reveal how the MTase recognizes their RNA substrates during N7 and 2'-O methylations. The proposed research is innovative and significant, because the flavivirus MTase is unique among all known MTases in which one MTase domain catalyzes two distinct N7 and 2'-O methylations in a viral RNA-dependent manner. The studies proposed here are expected to reveal complementary results to define the mechanism of the flavivirus MTase. The proposed work also fits well with the R21 funding mechanism: high risk but with significant outcome if accomplished. The knowledge acquired will significantly advance our understanding of flavivirus cap methylation, and in the long term, will lay the groundwork for designing inhibitors of the flavivirus MTase for antiviral therapy. PUBLIC HEALTH RELEVANCE: Many flaviviruses cause significant human diseases. Among then, dengue, yellow fever, West Nile, Japanese encephalitis, and tick-borne encephalitis are categorized as the NIAID Priority Pathogens. No effective antiviral therapy is currently available for treatment of flavivirus infections. We recently found that flavivirus methyltransferase (MTase) is a novel target for antiviral therapy. The current proposal is to define the molecular mechanism and function of the flavivirus MTase. These studies are expected to provide critical information for development of MTase therapies and vaccines against flaviviruses.